This invention relates to a method of inhibiting the oxidation of a polypeptide in a liquid or semi-solid medium, the polypeptide having an amino acid sequence comprising at least one methionine residue. More specifically, the invention relates to a method of inhibiting the oxidation of a tissue growth factor in a liquid or semi-solid medium, wherein the amino acid sequence of the tissue growth factor comprises at least one methionine residue. The invention further relates to stabilized, pharmaceutically effective preparations of such polypeptides.
In recent years, researchers have developed numerous techniques that have made possible the production and purification of various polypeptides on a commercial scale for therapeutic and pharmaceutical purposes. For example, polypeptides such as epidermal growth factor can be employed as the pharmacologically active component in ophthalmic preparations employed to enhance the repair of ocular tissue and also in cornea storage media employed in preserving eye tissue prior to surgical transplantation, i.e., keratoplasty. In particular, epidermal growth factor has been shown to have wound healing promoting activity (Plast. Reconstr. Surg., 64,766 (1979); J. Surg. Res., 33,164 (1982)); anti-inflammatory activity and analgesic activity (Japanese Laid-open Patent Publication No. 115785/1985). Epidermal growth factor is just one of a number of growth factors which are low molecular weight polypeptides that have the ability to stimulate the repair and maturation of tissue when applied thereto.
A growth factor can be selective with regard to both the type of tissue it acts upon as well as the extent of stimulation it causes in responsive cell types. For example, epidermal growth factor (EGF), vaccinia growth factor (VGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor alpha (TGF-.alpha.), transforming growth factor beta (TGF-.beta.), and insulin-like growth factor I (IGF-I) are all low molecular weight polypeptides which possess the ability to stimulate the division and maturation of certain cells.
The growth factors are known proteins, the properties and biological activities of which have been are described, for example, in review articles by Krisis et al., Biotechnology, February, 1985, pp. 135-140 and in Hormonal Proteins and Peptides, Ed. by Chao Hao Li, Vol 12, "Growth Factors" Academic Press (1984).
Epidermal growth factor is a low molecular weight protein (6040 daltons has been reported) previously isolated from mouse salivary glands according to the method of Savage and Cohen, J. Biol. Chem., 1972; 257; 7609-11. European Patent Office publication number EP 177,915 teaches the production of recombinant human EGF (rhEGF) by E. coli transformed with a vector containing DNA encoding EGF.
A process for obtaining transforming growth factor alpha is described in J.E. DeLarco and G.E. Todaro, "Growth Factors From Marine Carcoma Virus-Transformed Cells", Proc. Natl. Acad. Sci. U.S.A., 75:4001-4005, 1978.
EP 219,814 teaches the production of recombinant human insulin-like growth factor. I.E. Rinderknecht and R.E. Humbel teach the production of insulin-like growth factor I in "Polypeptides with the Non-Suppressible Insulin-Like and Cell-Growth Promoting Activities in Human Serum: Isolation, Chemical Characterization and Some Biological Properties of Forms I and II", Proc. Natl. Acad. Sci. U.S.A., 73:2365-2369, 1976.
Vaccinia growth factor is obtained from vaccinia virus-infected cells according to the methods of D.R. Twardzik, J.P. Braun, J.E. Ranchalis, G.E. Todaro and B. Moss, "Vaccinia Virus-Infected Cells Release A Novel Polypeptide Functionally Related To Transforming and Epidermal Growth Factors", Proc. Natl. Acad. Sci. U.S.A., 82:5300-5304, 1985.
Pharmaceutical preparations containing growth factors in an aqueous medium, such as ophthalmic preparations of EGF, commonly are packaged in plastic containers made of low density polyethylene (LDPE) or polypropylene for convenient storage and application. However, these plastic containers are readily permeable to oxygen. The oxygen causes rapid oxidation of the methionine residue(s) in the growth factor to methionine sulfoxide. It is the side chain of the methionine residue which is particularly vulnerable to oxidation (Manning et al., Pharmaceutical Research, Vol. 6, No. 11, 1989). Although the growth factor is still biologically active after its methionine residues have been oxidized to methionine sulfoxide, the growth factor is not acceptable for pharmaceutical use according to the standards of regulatory agencies, such as the FDA, when high levels of methionine sulfoxide are present. Current precautionary procedures to try to exclude oxygen during the manufacture and packaging of the preparation have proven to be ineffective in preventing significant methionine oxidation. The result is that the pharmaceutical preparation has a shorter effective life than is potentially possible if the oxidation reaction could be inhibited. In addition to growth factors, oxidation has been observed in many methionine containing peptide hormones during their isolation, synthesis and storage. Examples of some of the polypeptide hormones include adrenocorticotropic hormone, human growth hormone or somatotropin and the like.
Certain amino acids and various combinations thereof and surfactants, such as polysorbate and poloxamer and the like have been used to stabilize peptide and protein compositions. See, for example, Yu-Chang, John Wang and Musetta A. Hansen, "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers", Journal of Parenteral Science and Technology, 42:S14, 1988. However, none of the amino acids or surfactants are used to deter the oxidation of methionine residues to methionine sulfoxide in a liquid or semi-solid medium.
Therefore, there is a need for a method of inhibiting the oxidation in pharmaceutical vehicles of polypeptides having an amino acid sequence which comprises at least one methionine residue.